Colorectal cancer is second leading cause of cancer-related deaths among men and women in the United States, with metastasis to secondary organs (e.g., liver) playing a major role in patient mortality. Patients with metastatic, or stage IV, colon cancer display a 5 year survival rate of only 11%. Recent clinical and gene profiling studies indicate that a loss of Absent in Melanoma 2 (AIM2) expression in colon tumors is highly correlated with stage IV colon cancer and reduced patient survival. AIM2 is a cytosolic innate immune sensor that forms a multi-protein complex termed the inflammasome following binding of double-stranded DNA. I have recently reported that AIM2 suppresses colon cancer development independently of its inflammasome function by limiting the uncontrolled replication of colonic epithelial cells by acting as a checkpoint of Akt-mediated survival. Mechanistically, AIM2 suppresses Akt activity by targeting the PI3K family member DNA-dependent protein kinase (DNA-PK). Although DNA-PK activation and expression is reportedly elevated in colon tumors, and DNA-PK promotes Akt activation, cell survival and metastatic gene profiles, the function of DNA-PK during colon cancer pathogenesis is largely understudied. In addition, AIM2 has been suggested to limit tumorigenesis by regulating the composition of the intestinal microbiota, yet there is no direct evidence for this, and it is unknown if microbiota-derived DNA facilitates AIM2's tumor suppressor function or what AIM2-related factors are responsible for limiting tumorigenesis. In this proposal, I will test the hypothesis that AIM2 restricts cancer initiation and metastasis by limiting Akt and DNA-PK activation in response to the microbiota using the following specific aims: 1) Determine the functional requirement of DNA binding to AIM2 during the suppression of cancer-relevant pathways in vitro; 2) Assess the ability of AIM2 to limit DNA-PK-mediated colon cancer development and metastasis in vivo; and 3) Elucidate the mechanism by which AIM2 responds to and regulates the intestinal microbiota to limit colon cancer. This proposal builds upon my prior work that includes a strong background in cell signal transduction, intestinal microbiome profiling and animal models of colon cancer initiation while working in the laboratory of Jenny P-Y Ting at the Lineberger Comprehensive Cancer Center at the University of North Carolina at Chapel Hill. With additional technical assistance and core facility support from my collaborators, this K22 award will provide critical training in in vitro primary colon organoid culture systems, Crispr/Cas9 gene editing, mouse models of spontaneous tumorigenesis and metastasis, gene expression and signal transduction profiling of human cancer tissue and manipulation of the microbiome to target cancer, which are required to achieve my long-term goal of becoming an established tenured-track principle investigator in the area of Cancer Immunology. The proposed research will lead to a greater understanding of how the tumor suppressor AIM2 limits tumorigenesis and may lead to the development of novel therapeutics for the treatment of multiple cancers.